Cell culture incubators with integrated cell manipulation systems

ABSTRACT

Aspects of the invention relate to automated cell culture incubators. In some embodiments, automated cell culture incubators comprise an integrated manipulation device having one or more cell scrapers.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.provisional application U.S. Ser. No. 62/141,191, filed Mar. 31, 2015,entitled “Cell Culture Incubators With Integrated Cell ManipulationSystems”, the entire contents of which are incorporated by referenceherein.

FIELD

Aspects relate to cell culture incubators and to methods for using suchincubators.

BACKGROUND

Cell culture is a useful technique in both research and clinicalcontexts. For example, culture of mammalian cells is often performed inorder to establish clonal cell lines, tissue preparations, in vitrofertilization preparations or to expand populations of stem cells.However, maintenance of cell cultures in presently available cellincubators is a laborious process requiring highly trained personnel andstringent aseptic conditions. For example, to passage adherent cells atan appropriate degree of confluence, the adherent cells must bedissociated from the cell culture vessel in which they have beenpropagated and to which they have adhered. Typically, this processinvolves removing the cell culture vessel from a controlled environment(e.g., an incubator) to a cell culture hood and manually passaging thecells. This high level of human involvement can introduce contaminantsinto the culture or damage the cells, thereby lowering cultureefficiency and repeatability.

SUMMARY

Presently available cell culture incubators impose several barriers toproductive long-term cell culture. For example, many presently availablecell incubators require the removal of culture plates from the incubatorfor manipulating cells. Generally, removing cell culture plates from anincubator increases the threat of contamination to the culture becauseupon removal, the culture is exposed to non-aseptic conditions and/orvariations of the physical environment (e.g., changes in temperature,humidity, etc., or any combination thereof).

The instant document provides a cell culture system with an integratedmanipulation device, e.g., having one or more cell scrapers, that canreduce the exposure of cultures to contaminants, the externalenvironment and/or variations of the incubator environment byeliminating the need to remove culture vessels from the incubator formaintenance of cell cultures (e.g., passaging the cells).

Accordingly, in some aspects this document provides a cell cultureincubator comprising: an incubator cabinet comprising an internalchamber for incubation of cells in one or more cell culture vessels,(e.g., wherein the internal chamber is configured to hold the one ormore cell culture vessels); an external door opening from an externalenvironment to the internal chamber; an imager (e.g., an imager and animaging location), configured for imaging the cells within the internalchamber (e.g., when the one or more cell culture vessels are at theimaging location); a manipulator (e.g., a manipulator and a manipulatinglocation) having one or more cell scrapers for manipulating the cells(e.g., dissociating adherent cells from the cell culture vessel,cleaning a cell culture vessel) in the one or more cell culture vesselswithin the internal chamber; and a cell culture vessel transfer devicefor moving the one or more cell culture vessels between locations withinsaid internal chamber (e.g., from the imaging location to themanipulating location or from the manipulating location to the imaginglocation).

In some embodiments, the manipulator comprises one or more cellscrapers. In some embodiments, each cell scraper comprises a handleportion comprising a elongate member extending from a proximal regionthat is attachable or connectable with a manipulator base and a distalregion that comprises a scraping edge. In some embodiments, each cellscraper comprises a contiguous structure (e.g., a molded structure)comprising a scraping edge. In some embodiments, each cell scrapercomprises one or more interconnected parts. In some embodiments, eachcell scraper comprises a handle having an interface for replaceablyconnecting a scraping edge assembly to the handle. Thus, in someembodiments, a disposable scraper edge is provided that is detachable orreleasable from the scraper handle. In some embodiments, each cellscraper comprises a scraper edge contactable with the surface of a cellculture vessel and configured for scraping cells adhering to the surfacewithout substantially killing the cells. In some embodiments, theincubator further comprises a controller configured for controlling themanipulator to modulate the contact pressure between a scraping edge ofthe cell scraper and the surface of a cell culture vessel. In someembodiments, the incubator further comprises a sensor connected to thecell scraper (e.g., a strain gauge sensor) that provides signal to acontroller informative of a sensed pressure between a scraping edge ofthe cell scraper and the surface of a cell culture vessel, wherein thecontroller is configured to transmit a control signal to the manipulatorto increase or decrease the pressure between a scraping edge of the cellscraper and the surface of a cell culture vessel in response to thesensed pressure. In some embodiments, each cell scraper is readilyremovable from the manipulator. In some embodiments, each cell scraperis configured to perform scraping and liquid handling functions. In someembodiments, each cell scraper comprises a scraping edge configured toallow a definable range of scraping edge deflection on contact with acell culture vessel. In some embodiments, each cell scraper comprisesone or more components formed from a polymer. In some embodiments, eachcell scraper further comprises an opening configured for aspiratingcells and/or cell culture media, wherein the opening is positioned inclose proximity to a scraping edge.

In some embodiments, the cell scraper is disposable. In someembodiments, the cell scraper is configured to perform scraping andliquid handling functions. In some embodiments, the cell scrapercomprises a scraping edge configured to allow a definable range ofscraping edge deflection on contact with a cell culture vessel. In someembodiments, a scraping edge is formed from a polymer. In someembodiments, the edge comprises a geometry configured for excision ofcertain cells (e.g., pre-differentiated cells) from a larger populationof cells (e.g., healthy stem cell colonies). In some embodiments, thescraping edge further comprises an opening (e.g., an orifice) configuredfor transporting (e.g., aspirating, depositing, or aspirating anddepositing) cells and or cell culture media, wherein the opening (e.g.,orifice) is positioned in close proximity to the scraping blade.

In some embodiments, an opening (e.g., orifice). In some embodiments,the opening is configured for transporting (e.g., aspirating,depositing, or aspirating and depositing) cells and/or cell culturemedia and forms part of (e.g., is connected to) a channel. In someembodiments, a channel is integrated into a cell scraper (e.g., runningalong the inside or outside of a cell scraper handle and/or scrapingblade). In some embodiments, the incubator further comprises one or moreadditional manipulators, each having at least one cell scraper. In someembodiments, the at least one cell scraper (e.g., of the one or moremanipulators) is at least 2, 3, 4, 5, 10, 15, 20, 50, 100, 200, 300,500, or up to 1000 cell scrapers.

In some embodiments, the cell culture incubator further comprises acontroller of the manipulator for manipulating the cells. In someembodiments, the controller of the manipulator is configured to quantifyand modulate the contact force between the scraping edge and the surfaceof a cell culture vessel to which cells are adhered. In someembodiments, the controller is located exterior to the incubatorcabinet. In some embodiments, the controller is inside or integratedinto the incubator cabinet. In some embodiments, the controllercomprises a computer.

In some embodiments, an imager is provided in the incubator cabinet. Insome embodiments, the imager is configured to enable selective scrapingof cells with the manipulator while imaging the cells to be scraped orthat are scraped. In some embodiments, the imager is a holographicmicroscope. In some embodiments, the imager is a bright-fieldmicroscope. In some embodiments, the imager is a fluorescencemicroscope. In some embodiments, the imager is a phase-contrastmicroscope. In some embodiments, cell culture incubators furthercomprise a second imager, or a second imager and a third imager. In someembodiments, cell culture incubators having 3 imagers comprise aholographic microscope, a bright-field microscope, and a fluorescencemicroscope. In some embodiments, cell culture incubators having 3imagers comprise a phase-contrast microscope, a holographic microscope,and a fluorescence microscope. In some embodiments, the imager is usedto analyze the cells and determine automatically an area to be scraped.In other embodiments, the automatic selection of an area to be scrapedis modified by an operator. In other embodiment, the operator selectsmanually an area to be scraped from the plurality of images acquired bythe imagers.

In some embodiments, the one or more cell culture vessels are flasks,suspension culture flasks, spinner flasks, plates, petri dishes and/orbags. In some embodiments, the one or more cell culture vessels comprisefiducial marks for facilitating alignment of the one or more cellculture vessels to the imager and the manipulator.

In some embodiments, the manipulator for manipulating the cells is acell picker. In some embodiments, the cell culture incubator furthercomprises a controller of the manipulator for manipulating the cells. Insome embodiments, when the one or more cell culture vessels are movedfrom an imaging location to a manipulating location or from amanipulating location to an imaging location, the one or more cellculture vessels are substantially aligned.

In some aspects, this document provides a cell culture incubatorcomprising: an incubator cabinet comprising an internal chamber forincubation of cells in one or more cell culture vessels, wherein theinternal chamber is configured to hold the one or more cell culturevessels; a door opening to the internal chamber; a holographic imagercomprising a first imaging location, the holographic imager configuredfor imaging the cells within the internal chamber when the one or morecell culture vessels are at the first imaging location; a second imagercomprising a second imaging location, the imager configured for imagingthe cells within the internal chamber when the one or more cell culturevessels are at the second imaging location; a manipulator formanipulating the cells in the one or more cell culture vessels at thesecond imaging location; and a cell culture vessel transfer device formoving the one or more cell culture vessels from the first imaginglocation to the second imaging location or from the second imaginglocation to the first imaging location.

In some embodiments, the holographic imager is a holographic microscope.In some embodiments, the second imager is a bright-field microscope. Insome embodiments, the second imager is a fluorescence microscope. Insome embodiments, cell culture incubators further comprise a thirdimager. In some embodiments, cell culture incubators comprise threeimagers. In some embodiments, cell culture incubators having threeimagers comprise a holographic microscope, a bright-field microscope,and a fluorescence microscope.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures may be represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. Various embodiments of the invention will now be described, byway of example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic of an illustrative embodiment of a cell cultureincubator having an imager and a manipulator;

FIGS. 2A-2B are schematics of illustrative embodiments of cell cultureincubators; FIG. 2A shows a schematic of a cell culture incubator havinga second imager; FIG. 2B shows a schematic of a cell culture incubator,wherein the imaging location and the manipulating location are the samelocation; and,

FIG. 3 is a schematic depicting further components of cell cultureincubators.

FIGS. 4A-4D are schematics of illustrative embodiments of cell cultureincubators; FIG. 4A shows a schematic of a cell culture incubator havinga manipulator comprising a cell scraper; FIG. 4B shows a schematic of amanipulator comprising a plurality of cell scrapers; FIG. 4C is aschematic of two manipulators, each manipulator comprising a cellscraper; FIG. 4D is a schematic depiction of the range of motion alongx-, y- and z-axes of a manipulator comprising a cell scraper.

FIGS. 5A-5B are schematics of illustrative embodiments of cell scrapers.FIG. 5A shows a schematic of a cell scraper having a proximal end and adistal end comprising a scraper blade. FIG. 5B shows a schematic of acell scraper having a scraper blade comprising an opening and a channel(e.g., for aspirating cells and/or cell culture media).

FIGS. 6A-6B are schematics of illustrative embodiments of cell scrapers.FIG. 6A shows a side view schematic of a cell scraper having a proximalend and a distal end comprising a scraper blade. FIG. 6B shows a sideview schematic of a cell scraper having a scraper blade comprising anopening and a (e.g., channel for aspirating cells and/or cell culturemedia).

FIGS. 7A-7B are schematics of illustrative embodiments of cell scrapers.FIG. 7A shows a side view schematic of a cell scraper having a proximalend and a distal end comprising a scraper blade. FIG. 7B shows a sideview schematic of a cell scraper having a scraper blade comprising anopening and a (e.g., channel for aspirating cells and/or cell culturemedia).

FIGS. 8A-8B are schematics of illustrative embodiments of cell scrapers.FIG. 8A shows a back view (e.g., proximal to distal) schematic of a cellscraper having a proximal end and a distal end comprising a scraperblade. FIG. 8B shows a back view (e.g., proximal to distal) schematic ofa cell scraper having a scraper blade comprising an opening and achannel (e.g., for aspirating cells and/or cell culture media).

FIGS. 9A-9B are schematics of illustrative embodiments of cell scrapers.FIG. 9A shows a schematic of a cell scraper having a proximal end and adistal end comprising a scraper blade. FIG. 9B shows a schematic of acell scraper having a scraper blade comprising an opening and a channel(e.g., for aspirating cells and/or cell culture media).

FIGS. 10A-10B are schematics of illustrative embodiments of cellscrapers. FIG. 10A shows a schematic of a cell scraper having a proximalend and a distal end comprising a scraper blade. FIG. 10B shows aschematic of a cell scraper having a scraper blade comprising an openingand a channel (e.g., for aspirating cells and/or cell culture media).

FIG. 11 shows a schematic of a cell scraper having a proximal end and adistal end comprising a scraper blade.

FIG. 12 shows a schematic of a cell scraper having a scraper bladecomprising an opening and a channel (e.g., for aspirating cells and/orcell culture media).

FIGS. 13A-13C are schematics of illustrative embodiments of cellscrapers having a proximal end and a distal end comprising a scraperblade.

FIG. 14 is a schematic of a manipulator comprising a cell scraper with adepiction of the range of motion along x-, y- and z-axes of amanipulator comprising a cell scraper.

FIGS. 15A-15C are schematics of illustrative embodiments of cellscrapers. FIG. 15B shows a front view schematic of a scraper blade. FIG.15C shows a side view schematic of a scraper blade.

DETAILED DESCRIPTION

Currently used cell culture incubators impose barriers to the success ofcell cultures. For example, many cell culture incubators require theremoval of cell culture vessels and their subsequent manual handling forimaging and manipulating of the cultured cells. Removal of culturedcells from the protected environment provided by the incubator increasesexposure of the culture to potential contaminants and to environmentalchanges that may interfere with cell growth. In some cases, removal ofcultures from the incubator exposes laboratory personnel to pathogenicorganisms being cultured in the incubator. Furthermore, manual handlingof cultures by human operators introduces the possibility ofcontamination introduced by human error, such as improper steriletechnique. This document provides cell culture incubators having anintegrated manipulator device (e.g., incubators comprising a cellscraper).

In some aspects, this document relates to a cell culture incubatorhaving: an incubator cabinet comprising an internal chamber forincubation of cells in one or more cell culture vessels, wherein theinternal chamber is configured to hold the one or more cell culturevessels; an external door opening from an external environment to theinternal chamber; an imager and an imaging location, the imagerconfigured for imaging the cells within the internal chamber when theone or more cell culture vessels are at the imaging location; amanipulator and a manipulating location for manipulating the cells inthe one or more cell culture vessels within the internal chamber; and acell culture vessel transfer device for moving the one or more cellculture vessels from the imaging location to the manipulating locationor from the manipulating location to the imaging location. In someembodiments, the manipulator comprises one or more cell scrapers.

As used herein, an “incubator cabinet” is a housing that includes one ormore chambers configured to hold one or more cell culture vessels. Insome embodiments, an incubator cabinet includes a transfer chamber andan internal chamber, one or both of which are configured to hold one ormore cell culture vessels. In some embodiments, an incubator may includeone or more other elements such as one or more gas sources (e.g., acompressed gas cylinder or ozone generator), tubing (e.g., to convey oneor more liquids or gases such as water, distilled water, deionizedwater, cell culture medium, air, carbon dioxide, ozone, and oxygen),airflow mechanisms (e.g., valves, release valves, pinholes, gasregulators, and mass flow regulators), pressure mechanisms (e.g., a pumpsuch as a dry scroll pump, rotary pump, momentum transfer pump,diffusion pump, or diaphragm pump; a suction tube; a vacuum system; andan air blower), environmental monitors and controls (e.g., a gas sensorand/or monitor to sense and/or control concentrations of gases such ascarbon dioxide, oxygen, and ozone; heat sources or sinks; temperaturemonitors and controls; humidity monitors; gas scrubbers; air filters;instrumentation for measuring particulate matter; pressure gauges; andflow meters), doors (e.g., openings or panels) windows (e.g., opticalwindows made of glass, plastic, composite, or other substantiallytransparent material for viewing an area inside the incubator cabinet),ports (e.g., to permit the introduction or removal of one or more gasesor liquids), light sources (e.g., lamps, bulbs, lasers, and diodes),optical elements (e.g., microscope objectives, mirrors, lenses, filters,apertures, wave plates, windows, polarizers, fibers, beam splitters, andbeam combiners), imaging elements (e.g., barcode readers, cameras,etc.), electrical elements (e.g., circuits, cables, power cords, andpower supplies such as batteries, generators, and direct or alternatingcurrent supplies), computers, mechanical elements (e.g., motors, wheels,gears, robotic elements, and actuators such as pneumatic actuators,electromagnetic actuators, motors with cams, piezoelectric actuators,and motors with lead screws), and control elements (e.g., spin-wheels,buttons, keys, toggles, switches, cursors, screws, dials, screens, andtouch-screens). In some embodiments, one or more of these other elementsare part of the incubator, but are external to the incubator cabinet. Insome embodiments, one or more of these other elements are includedwithin the incubator cabinet.

As used herein, an “internal chamber” is a chamber disposed in anincubator cabinet. An internal chamber may include one or more windows(e.g., optical windows made of glass, plastic, composite, or othersubstantially transparent material for viewing an area inside theincubator cabinet). An internal chamber may include at least one door(e.g., for permitting the transfer of items into or out of the internalchamber). In some embodiments, the at least one door may be disposedbetween the internal chamber and a transfer chamber. In certainembodiments, an interlock may prevent the door from opening at anundesirable time (e.g., when a portion of the incubator cabinet is opento the surrounding environment so that contaminants cannot enter theinternal chamber). An internal chamber may be of any appropriate sizeand geometry. In some embodiments, an incubator cabinet may include morethan one internal chamber. In other embodiments, an internal chamber mayinclude one or more partitions to define different regions of aninternal chamber. One or more internal chambers or partitions thereofmay have different environmental conditions. The environment (e.g., airpressure, gas content, temperature, light, and humidity) inside aninternal chamber may be measured and/or controlled by one or moremeters, monitors, sensors, controls, pumps, valves, apertures, and/orlight sources. In some embodiments, an internal chamber may have agas-tight or hermetic seal, e.g., around one or more windows or doors.In particular embodiments, sealants such as grease and/or mechanicalelements such as o-rings, gaskets, septa, KF, LF, QF, quick coupling, orother sealing mechanisms may be used to establish one or more gas-tightseals. In some embodiments, grooves, depressions, protrusions, and/ormolded plastic elements may facilitate in establishing one or moregas-tight seals.

An internal chamber may be made of any useful material. In someembodiments, an internal chamber may include one or more plastics,polymers, metals, or glasses.

As used herein, a “door” is an element that permits communicationbetween two or more environments or regions when opened and preventscommunication between the two or more environments or regions whenclosed. A door may be of any type, such as a sliding door, pocket door,swinging door, hinged door, revolving door, pivot door, or folding door.The door may be manually, mechanically, or electrically operated. Forexample, an operator may open or close a door by manually grasping,pulling, pushing, and/or otherwise physically interacting with the dooror an element thereof (e.g., a handle) or by operating a mechanicalcontrol (e.g., a button, toggle, spin-wheel, key, switch, cursor, screw,dial, screen, or touch-screen). In certain embodiments, a door may becontrolled by electrical or digital controls, such as by a computer. Adoor may be an automatically opening door. For example, a door mayinclude a sensor, such as a pressure, infrared, motion, or remotesensor, that detects whether the door is open or closed and/or controlswhen the door opens or closes. A door may open by mechanical, pneumatic,electrical, or other means. In some embodiments, one or more doors mayinclude one or more locking mechanisms. In particular environments, oneor more doors may include one or more interlocks (e.g., a mechanicalinterlock such as a pin, bar, or lock or an electrical interlock such asa switch) to prevent one or more doors from opening at an undesirabletime (e.g., when one or more chambers are open to the outsideenvironment).

A transfer device for moving one or more items may be used to move itemsbetween the transfer chamber and the internal chamber. In someembodiments, the transfer device comprises a conveyor belt or othersimilar device for maneuvering items. Non-limiting examples of itemsthat can be moved by transfer devices include cell culture vessels,pipettes, containers, syringes and other materials and instrumentsutilized in the culture of cells. In some embodiments, more than onetransfer device may be included. In some embodiments, one or moretransfer devices are located in the transfer chamber and/or in theinternal chamber. In some embodiments, a transfer device may include oneor more robotic elements. For example, a transfer device may compriseinclude one or more robotic arms capable of gripping, lifting, pushing,grabbing, sliding, rotating, translating, releasing, raising, lowering,and/or tilting one or more items (e.g., pipettes).

In some embodiments, the transfer device is a cell culture vesseltransfer device. As used herein, a “cell culture vessel transfer device”refers to a device that can transfer one or more cell culture vesselsfrom a first location to a second location. In some embodiments, thetransfer device is anchored within the internal chamber. In certainembodiments, the transfer device may transfer one or more items to orfrom multiple locations in an incubator cabinet. For example, a cellculture vessel transfer device may be used to move a cell culture vesselfrom a transfer chamber to an internal chamber, and/or from a storagelocation to an imaging location. In some embodiments, an incubatorcabinet includes more than one transfer device for moving one or moreitems (e.g., separate means for transferring items between and withinchambers). A cell culture vessel transfer device may include one or moreelements such as valves (e.g., electromagnetic or pneumatic valves),gears, motors (e.g., electrical or stepper motors), stages (e.g., xy orxyz stages), pistons, brakes, cables, ball-screw assemblies,rack-and-pinion arrangements, grippers, arms, pivot points, joints,translational elements, or other mechanical or electrical elements. Insome embodiments, a cell culture vessel transfer device may include oneor more robotic elements. For example, a cell culture vessel transferdevice may include a robotic arm capable of gripping, lifting, pushing,grabbing, sliding, rotating, translating, releasing, raising, lowering,and/or tilting one or more cell culture vessels. In some cases, the cellculture vessel transfer device selectively and releasably grips one ormore cell culture vessels. In certain embodiments, a cell culture vesseltransfer device may include an arm coupled to a mechanical gripper. Forexample, an arm may include a mechanical gripper at or near one end forreleasably gripping a cell culture vessel and be securely coupled to asurface or element of the incubator at or near the other end. In someembodiments, a robotic arm includes a pivot point where the mechanicalgripper couples to the arm and one or more pivot and/or translationaljoints along the arm to permit flexible rotation and translation of aportion of the arm. In this manner, a robotic arm may access one or morecell culture vessels at different horizontal and vertical positionswithin an incubator cabinet (e.g., within a storage array in an internalchamber).

In some embodiments, a cell culture vessel transfer device is anautomated transfer device. For example, the automated transfer devicemay be a robotic arm controlled by a computer that is programmed to movecell culture vessels from a storage location within the internal chamberof the incubator to an imaging location within the internal chamber ofthe incubator. In some embodiments, a cell culture vessel transferdevice is manually operated. For example, a robotic arm located insidethe internal chamber of an incubator may be operated by auser-controlled joystick from a location outside of the internal chamberof the incubator in order to move cell culture vessels from a storagelocation within the internal chamber of the incubator to an imaginglocation within the internal chamber of the incubator.

As used herein, a “cell culture vessel” is a device including a housingand one or more chambers for culturing cells. In some embodiments, thehousing is a frame. The frame may be coupled to a lid. The one or morechambers may include cell culturing media including one or moremembranes. In some embodiments, a cell culture vessel may includenutrients for promoting the growth of cells. In certain embodiments, acell culture vessel may entirely enclose one or more cells or groupsthereof. The housing of a cell culture vessel may include one or morepores or openings to permit the transfer of gases between a cell culturevessel and its surrounding environment. Non-limiting examples of cellculture vessels include flasks, suspension culture flasks, spinnerflasks, plates, petri dishes and/or bags. In certain embodiments, a cellculture vessel includes a transparent or optically clear window. Forexample, a lid coupled to the housing of a cell culture vessel mayinclude an optically clear portion for viewing cells e.g., with amicroscope or other imager. In some embodiments, a cell culture vesselincludes one or more portions that are substantially non-reflective. Insome embodiments, the cell culture vessel is barcoded. Therefore, insome embodiments, the incubator includes a barcode reader.

In some embodiments, cell culture vessels may be pre-kitted with one ormore reagents desired for a particular purpose, e.g., for growing cells,for differentiating cells, for subjecting cells to a particular assaycondition, etc. In some embodiments, pre-kitted cell culture vesselscontain reagents useful for performing a particular experiment (e.g.,cell growth media, growth factors, selection agents, labeling agents,etc.) on a cell culture, in advance of the experiment. Pre-kitted cellculture vessels may facilitate experimental protocols by providing cellculture-ready vessels that do not require the addition of reagents. Forexample, progenitor cells from a patient may be added to a cell culturevessel pre-kitted with reagents for cell differentiation for the purposeof expanding a population of differentiated cells for autologous celltherapy. Pre-kitted cell culture vessels can be stored at anyappropriate temperature, which is determined by the recommended storageparameters of the reagents within the pre-kitted cell culture vessel. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −80° C. and about 37° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −80° C. and about −20° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about −20° C. and about 4° C. Insome embodiments, pre-kitted cell culture storage vessels are storedprior to use at temperatures between about 4° C. and about 37° C. Insome embodiments, pre-kitted cell culture vessels are disposable. Insome embodiments, pre-kitted cell culture vessels are reusable and/orrefillable.

As used herein, a “storage location” refers to a location at which oneor more cell culture vessels is stored (e.g., within an incubatorcabinet). For example, one or more cell culture vessels may be stored ata storage location and later transferred to a different location (e.g.,an imaging location). The storage location may be disposed in theinternal chamber of the incubator cabinet. A storage location may beconfigured for storing a plurality of cell culture vessels. For example,a storage location may include one or more storage arrays, racks,shelves, pigeon-holes, cubbies, trays, slots, or other positions ormechanisms. In some embodiments, a storage location may be configured tostore cell culture vessels horizontally, while in other embodiments itmay configured to store cell culture vessels vertically. For example, astorage location may include a plurality of slots to receive cellculture vessels stacked vertically over one another. A storage locationmay be configured to hold 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 100, or any other number of cell culture vessels. Insome embodiments, a storage location may be configured to hold greaterthan 100 cell culture vessels. In some embodiments, a storage locationmay include a mechanism for moving one or more storage arrays, racks,shelves, pigeon-holes, cubbies, trays, slots, or other positions ormechanisms. For example, a storage location may include one or moremotors and movable stages (e.g., an xy or xyz stage) to move a storagerack from one position in an internal chamber to another position in aninternal chamber, e.g., to facilitate access to one or more cell culturevessels stored in different locations. In some embodiments, theincubator cabinet may include one or more cell culture vessel transferdevices for moving one or more cell culture vessels.

A storage location may be configured to securely hold or receive one ormore cell culture vessels. For example, one or more components of thestorage location may include one or more locking mechanisms that haveone or more adhesive, magnetic, electrical, and/or mechanical components(e.g., snaps, fasteners, locks, clasps, gaskets, o-rings, septa,springs, and other engagement members). In some embodiments, a storagelocation and/or cell culture vessel may include one or more grooves ordepressions and/or may involve pieces of molded plastic. For example, acell culture vessel may include one or more protruded features (e.g., arim or knob) that are molded for insertion into one or morecorresponding grooves, holes, or depressions at a storage location. Insome cases, a cell culture vessel may include one or more grooves,holes, or depressions that are molded to fit one or more correspondingprotruded features at a storage location.

As used herein, an “imager” refers to an imaging device for measuringlight (e.g., transmitted or scattered light), color, morphology, orother detectable parameters such as a number of elements or acombination thereof. An imager may also be referred to as an imagingdevice. In certain embodiments, an imager includes one or more lenses,fibers, cameras (e.g., a charge-coupled device camera or CMOS camera),apertures, mirrors, light sources (e.g., a laser or lamp), or otheroptical elements. An imager may be a microscope. In some embodiments,the imager is a bright-field microscope. In other embodiments, theimager is a holographic imager or microscope. In other embodiments, theimager is a fluorescence imager or microscope. In other embodiments, theimager is a phase-contrast microscope.

As used herein, a “fluorescence microscope” refers to an imaging devicewhich is able to detect light emitted from fluorescent markers presenteither within and/or on the surface of cells or other biologicalentities, said markers emitting light at a specific wavelength inresponse to the absorption a light of a different wavelength.

As used herein, a “bright-field microscope” is an imager thatilluminates a sample and produces an image based on the light absorbedby the sample. Any appropriate bright-field microscope may be used incombination with an incubator cabinet provided herein.

As used herein, a “phase-contrast microscope” is an imager that convertsphase shifts in light passing through a transparent specimen tobrightness changes in the image. Phase shifts themselves are invisible,but become visible when shown as brightness variations. Any appropriatephase-contrast microscope may be used in combination with an incubatorprovided herein.

As used herein, a “holographic imager” is an imager that providesinformation about an object (e.g., sample) by measuring both intensityand phase information of electromagnetic radiation (e.g., a wave front).For example, a holographic microscope measures both the lighttransmitted after passing through a sample as well as the interferencepattern (e.g., phase information) obtained by combining the beam oflight transmitted through the sample with a reference beam.

A holographic imager may also be a device that records, via one or moreradiation detectors, the pattern of electromagnetic radiation from asubstantially coherent source, diffracted or scattered directly by theobjects to be imaged, without interfering with a separate reference beamand with or without any refractive or reflective optical elementsbetween the substantially coherent source and the radiation detector(s).

In some embodiments, an incubator cabinet includes a single imager. Insome embodiments, an incubator cabinet includes two imagers. In someembodiments, the two imagers are the same type of imager (e.g., twoholographic imagers or two bright-field microscopes, or twophase-contrast microscopes). In some embodiments, the first imager is abright-field microscope and the second imager is a holographic imager.In some embodiments, an incubator cabinet comprises more than 2 imagers.In some embodiments, cell culture incubators comprise three imagers. Insome embodiments, cell culture incubators having 3 imagers comprise aholographic microscope, a bright-field microscope, and a fluorescencemicroscope. In some embodiments, cell culture incubators having 3imagers comprise a phase-contrast microscope, a holographic microscope,and a fluorescence microscope.

As used herein, an “imaging location” is the location where an imagerimages one or more cells. For example, an imaging location may bedisposed above a light source and/or in vertical alignment with one ormore optical elements (e.g., lens, apertures, mirrors, objectives, andlight collectors).

As used herein, a “fiducial mark” refers to a feature that facilitatesalignment of one or more components. In some embodiments, fiducial marksmay include one or more hole apertures over a fluorescent media orprinted or embossed fluorescent material. In other embodiments, fiducialmarks may include grids, lines, or symbols. In some embodiments, one ormore cell culture vessels include one or more fiducial marks tofacilitate alignment of one or more cell culture vessels with an imager.In some embodiments, fiducial marks may be associated with moving parts,including transfer devices and robotics devices.

In some embodiments, a cell culture vessel is substantially aligned withan imager. In some embodiments, a cell culture vessel is substantiallyaligned with an imager via the use of at least one fiducial mark. Asused herein, the term “substantially aligned” implies that one or moreelements are substantially overlapping, identical, and/or in line withone another. The substantial alignment of one or more cell culturevessels at one or more locations (e.g., imaging locations) mayfacilitate the analysis of a sample by permitting overlapping images ofthe cell culture vessel to be obtained. For example, a cell culturevessel may be imaged at a first imaging location by a first imager andsubsequently imaged at a second imaging location by a second imager. Ifthe imaging fields of the respective imagers are substantially aligned,the images recorded by the first and second imagers may be combined(“stitched together”) for analysis. One or more fiducial marks presenton one or more cell culture vessels may facilitate substantialalignment. In some cases, one or more fiducial marks present at one ormore imaging or other locations (e.g., manipulation or maintenancelocations) may facilitate substantial alignment.

As used herein, a “manipulator for manipulating cells” refers to adevice for manipulating cells in the internal chamber. The manipulatormay include one or more needles, capillaries, pipettes, and/ormicromanipulators. In some embodiments, a manipulator comprises one ormore cell scrapers. As used herein, “cell scraper” refers to a devicecomprising a scraping edge suitable for scraping cells off of a surface.In some embodiments, a cell scraper comprises a handle portioncomprising a elongate member extending from a proximal region that isattachable or connectable with a manipulator base and a distal regionthat comprises a scraping edge. In some embodiments, a cell scraper is acontiguous structure (e.g., a molded structure) comprising a scrapingedge. However, in some embodiments, a cell scraper comprises one or moreinterconnected parts. For example, in some embodiments, a cell scrapercomprises a handle having an interface for replaceably connecting orattaching a scraping edge or scraping edge assembly to the handle. Insome embodiments, the scraping edge is a portion of a cell scrapercontactable with the surface of a cell culture vessel or other surfaceand suitably configured for scraping matter from the surface forcleaning the surface and/or for scraping cells adhering to the surfacewithout substantially killing the cells, e.g., by mechanically lysingthe cells. In some embodiments, it is desirable for a scraping edge orscraping edge assembly to be disposable in order to preventcross-contamination between cell cultures. Thus, in some embodiments,the scraping edge or scraping edge assembly is disposable.

In some embodiments, a scraping edge comprises a blade, wiper or anotherwise substantially planar surface comprising an edge (e.g., abeveled edge) that is configured for removing cells from the surface ofa cell culture vessel when pushed or pulled along the surface of thecell culture vessel. In some embodiments, a scraping edge can be made ofa polymer or combination of polymers (e.g., plastic, silicone), glass,metal or any other suitable material. However, in some embodiments, thescraping edge comprises mechanical/material properties that allow for adefinable range of scraping edge deflection on contact, which allows forclose control of angle of contact with the cell culture vesselsurface/adherent cells. In some embodiments, edge of the cell scraper isformed from a polymer. Examples of polymers used to form scraping edgeinclude, but are not limited to, silicone, polyurethane, polyethylene,polyester, polypropylene, polybutylene, polystyrene, polyvinyl chloride(PVC), and nylon. In some embodiments, a blade comprises a geometryconfigured for excision of certain cells (e.g., pre-differentiatedcells) from a larger population of cells (e.g., healthy stem cellcolonies).

In certain embodiments, a cell scraper is configured to perform scrapingand liquid handling functions. For example, in some embodiments ascraper further comprises an opening(s) (e.g., orifice(s)) in closeproximity to a scraping edge (e.g., blade) together with an accompanyingport to a pipet head or other fluid movement device (e.g., a pump,vacuum chamber), thus allowing simultaneous scraping and aspiration ofcellular material. Such a configuration is useful, in some embodiments,for the option for cell cleaning or colony picking (e.g., in the contextof stem cells separation).

In some embodiments, an opening (e.g., orifice) configured fortransporting (e.g., aspirating, depositing, or aspirating anddepositing) cells and/or cell culture media forms part of a channel. Asused herein, a “channel” refers to a partially enclosed conduit (e.g., acylindrical, tubular, or cuboid passage) designed to allow the transportof a objects (e.g., cell culture media, cells) from one location toanother location. In some embodiments, a channel is integrated into acell scraper (e.g., running along the inside or outside of a cellscraper handle and/or scraping blade). For example, in some embodiments,a cell scraper comprises a hollow handle configured to contain a channelcomprising an opening, wherein the opening is located in or on thescraping blade (e.g., scraping edge) of the cell scraper. A channelresiding within the interior of a hollow scraper handle (e.g., in ascraper handle cavity) can have a volume ranging from about 0.1% of thevolume within the handle cavity to about 99% of the volume within thehandle. For example, a channel can be a separate capillary tube orhollow needle (e.g., a needle having a gauge between 28 gauge and 10gauge) that is directly connected or attached to a wall (e.g., runningalong an interior wall or exterior wall) of a hollow scraper handle. Insome embodiments, the entire volume within a hollow scraper handle formsa channel. For example, in some embodiments, a cell scraper comprises ahollow handle and a scraper blade having an opening which is connectedto the hollow interior (e.g., channel) of the handle. In someembodiments internal structures such as a mesh, grill, screen, orstar-shaped structure, to facilitate the mixing or breaking apart ofaggregated cells, are integrated into the channel of a cell scraperhandle and/or scraping blade. Without wishing to be bound by anyparticular theory, a cell scraper comprising an opening and a channel iscapable of simultaneously performing cell scraping and liquid handlingfunctions (e.g., simultaneously triturating cells and removing debris,such as triturated cells and cell culture media).

In some embodiments, a manipulator comprises at least one cell scraper.For example, a manipulator may comprise between about 1 and about 100,about 10 and about 100, about 20 and about 1000, or about 50 and about500 cell scrapers. In some embodiments, a manipulator comprises 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 24, 48, 96, 384, or 1536 cell scrapers. Insome embodiments, a manipulator having a plurality of cell scrapers isreferred to as having a “bank” of cell scrapers, as shown in FIG. 4B. Insome embodiments, a manipulator comprises a single cell scraper. In someembodiments, an incubator may comprise multiple manipulators, eachmanipulator having at least one cell scraper (e.g., as depicted in FIG.4C).

In some embodiments, cell scrapers are useful for dissociating adherentcells from the surface (or surfaces) of a cell culture vessel. Forexample, cell scrapers may be utilized for manual, mechanical looseningof protein bonds of adherent cells to a culture vessel. In someinstances, mechanical dissociation by scraping is aided by the use of anenzyme such as trypsin or ACCUTASE®. However, in certain embodiments,cell scraping is the preferred method of cell dissociation, primarilybecause the use of enzymes as the primary means of dissociation requiresrelatively long exposure of the cells to the enzyme in order to insuresufficient cellular detachment from the substrate. In some embodiments,scraping is the preferred methods for passaging stem cells duringexpansion.

A manipulator may include a cell picker. A manipulator for manipulatingcells may operate by detecting desirable cells or groups thereof presentat a first location based on a predetermined criterion and transferringthe desired cells or groups thereof from the first location to a secondlocation. A cell picker may detect, pick, and/or transfer desirable orundesirable (e.g., pre-differentiated cell weeding) cells or groupsthereof based on a manual or automated analysis. In some embodiments,information produced by an imager may be analyzed to detect desirable orundesirable cells. The cell picker may then transfer the desirable orundesirable cells to the second location. For example, an imager mayimage cells in or on a cell culture vessel at an imaging location, andthe image used to identify desirable or undesirable cells or groupsthereof. The cell picker may then transfer the desirable or undesirablecells, e.g., by contacting each desired cell or cells with a needle,capillary, pipette, or micromanipulator and effecting a movement of thecell or cells, from their first location to a second location in or onthe cell culture vessel or elsewhere in the internal chamber. In someembodiments, the first location of the cells may be in or on a cellculture vessel. In particular embodiments, a cell picker transfers cellsfrom a first location in or on a cell culture vessel to a secondlocation on the same cell culture vessel. In other embodiments, a cellpicker transfers cells from a first location in or on a first cellculture vessel to a second location in or on a second cell culturevessel. In certain other embodiments, a cell picker transfers cells froma first location in or on a cell culture vessel to a second location inthe internal chamber that is not in or on a cell culture vessel.

In some embodiments, the manipulator includes at least onemicroelectrode. As used herein, the term “microelectrode” refers to anelectrical conductor used to deliver electrical stimulation to a cell.For example, microelectrodes can be used to deliver genetic materialinto a cell by electroporation. In some embodiments, the manipulatorincludes at least one microinjector. Generally, microinjectors are glassmicropipettes that have been pulled to form a sharp, hollow structurecapable of piercing the membrane of a cell and serving as a conduit forthe introduction of genetic material into the cell.

In some embodiments, a manipulator is manually operated. For example, amanipulator having a cell picker located inside the internal chamber ofan incubator cabinet may be electronically-linked to and controlled by auser-directed joystick located outside the internal chamber of theincubator cabinet. In some embodiments, the user-directed joystick isconnected to a display device. In some embodiments, the display deviceshows images captured by an imaging device inside the internal chamberof the incubator cabinet.

In some embodiments, a manipulator is automated. For example, amanipulator inside an internal chamber of an incubator cabinet may beelectronically connected to a controller outside of the incubatorcabinet, which is electronically connected to a computer that directsthe manipulator. In some embodiments, a controller interfaces withhardware configured for quantifying and modulating contact force betweena scraping edge and the surface of the cell culture vessel to which thecells are adhered. For example, a manipulator or cell scraper mayfurther comprise a sensor (e.g., a pressure sensor) that provides signalto a controller informative of a sensed pressure, which in response tothe controller transmits a signal (e.g., to a manipulator) to increaseor reduce the pressure which the manipulator exerts upon the scrapingedge (e.g., a blade) of the cell scraper. In some embodiments, acontroller comprises software and/or hardware configured for programmingup to 360° rotation of the scraper tip/blade, thus allowing forprogramming of scraping motion in coordination with linear motion in thex-, y-. and z-axis such that a constant angle of attack is maintainedbetween the scraper blade and the cells being scraped, regardless ofculture vessel well geometry. In some embodiments, the controllerinterfaces with one or more components or hardware configured to permitlocation and determination, e.g., via imaging, of aggregated clump(s) ofcells, thus allowing subsequent scraping/aspiration via themanipulator-controlled cell scraper.

One or more elements of the manipulator for manipulating cells may besterilized, for example using a sterilizing composition or method (e.g.,ethanol or ozone gas, UV Light, Hydrogen peroxide), prior tomanipulation.

As used herein, “manipulation location” refers to the location at whichcells are manipulated by a manipulator for manipulating cells (e.g., acell picker). In certain embodiments, the manipulation location may bethe same as the imaging location.

According to one aspect, the cell culture incubator includes anincubator cabinet with an imaging location and a manipulating location.Cells of a cell culture vessel are imaged at the imaging location by animager and manipulated at the manipulating location by a manipulator. Insome embodiments, the imaging location and the manipulating location aretwo distinct locations within the incubator cabinet. The cell cultureincubator may include a transfer device that moves cell culture vesselsbetween the imaging location and the storage location. In otherembodiments, the imaging location and the manipulating location are thesame, such that the cells of culture vessels are imaged at themanipulation location.

In some embodiments, an imager may be used in conjunction with amanipulator. For example, an imager may image cells in or on a cellculture vessel at an imaging location, and the image used to identifydesirable cells or groups thereof. The cell picker, which may or may notbe resident at the imaging location, may then transfer the desirable orundesirable cells, e.g., by contacting each desired cell or cells with aneedle, capillary, pipette, or micromanipulator and effecting a movementof the cell or cells, from their first location to a second location inor on the cell culture vessel or elsewhere in the internal chamber. Insome embodiments, a cell picker transfers cells from a first location inor on a cell culture vessel to a second location on the same cellculture vessel. In other embodiments, a cell picker transfers cells froma first location in or on a first cell culture vessel to a secondlocation in or on a second cell culture vessel. In certain otherembodiments, a cell picker transfers cells from a first location in oron a cell culture vessel to a second location in the internal chamberthat is not in or on a cell culture vessel.

In some embodiments, a single location within the incubator cabinet mayserve as an imaging location and a manipulating location. In oneembodiment, cells are imaged as they are manipulated by the manipulator.In some embodiments, the imaging location and imaging location may be atseparate locations within the incubator cabinet.

In some embodiments, the manipulator includes sensors that allow it toreport its position and determine when it has touched the bottom of thecell culture vessel. In some embodiments, an imager may be used to guidethe manipulator in order to achieve repeatability and accuracy. In someembodiments, compliance (e.g., springiness) in the manipulator may beused to relax the need for extreme mechanical accuracy.

Turning to the figures, FIG. 1 depicts one illustrative embodiment of acell culture incubator. The cell culture incubator includes an incubatorcabinet having an internal chamber (100) for incubation of cells in oneor more cell culture vessels. The incubator cabinet includes an externaldoor (101) that opens and closes to permit communication between anexternal environment and the incubator cabinet. In some embodiments, theexternal door opens and closes to permit communication between anexternal environment and the internal chamber. The internal chamber isconfigured to hold one or more cell culture vessels. The one or morecell culture vessels are stored in a storage location (102). In someembodiments, the storage location is a free-standing structure. Forexample, a storage location may be a test tube or culture flask rackthat can be removed from the internal chamber of the incubator forloading and unloading of culture vessels. In some embodiments, thestorage location is affixed to a surface of the internal chamber. Forexample, the storage location may be a series of racks or shelves thatare connected to the walls or floor of the internal chamber and are thusnot able to be removed from the incubator cabinet.

In some embodiments, the cell culture incubator includes a cell culturevessel transfer device (103) for moving one or more cell culturevessels. The cell culture transfer device may be affixed to anyappropriate surface of the internal chamber of the incubator. Forexample, the cell culture vessel transfer device may be affixed to thetop or ceiling of the internal chamber. Alternatively, the cell culturevessel transfer device may be affixed to a side wall of the internalchamber. In some embodiments, the cell culture vessel transfer device isnot affixed to the wall of the internal chamber. For example, the cellculture vessel transfer device may rest on a wheeled tripod or othermobile structure that can be moved around the internal chamber.

In some embodiments, the transfer device moves one or more cell culturevessels from a storage location (102) to an imaging location (105) or toa manipulation location (107). The transfer device (103) can also moveone or more cell culture vessels from an imaging location (105) to amanipulation location (107) or from a manipulation location (107) to animaging location (105). When imaging or manipulation are complete, thetransfer device (103) moves one or more cell culture vessels from animaging location (105) or a manipulation location (107) to a storagelocation (102).

In some embodiments, the incubator cabinet includes a first imaginglocation (105) and a manipulation location (107). In some embodiments,one or more imaging locations are located on a surface of the internalchamber opposite from an imager. In some embodiments, imaging locationsare platforms, either free-standing or affixed to a surface of theinternal chamber. In some embodiments, the platform is movable. Forexample, a movable platform may be affixed to two or more rods thatallow the platform to be moved left, right, forward, backward, up ordown in relation to an imager. In some embodiments, the movable platformis motorized.

In some embodiments, the incubator cabinet includes a first imager (104)that images the cells of cell culture vessels when the vessels are atthe first imaging location (105). In some embodiments, the first imageris a bright-field microscope. In some embodiments, the first imager is aholographic microscope. In some embodiments, the first imager is aphase-contrast microscope.

In some embodiments, a manipulator (106) manipulates the cells of cellculture vessels when the vessels are at the manipulation location (107).In some embodiments, the manipulator has an array of needles,capillaries, pipettes, and/or micromanipulators. For example, themanipulator may include a cell picker. In some embodiments, amanipulator comprises one or more cell pickers. In some embodiments, themanipulator may include a cell scraper. In some embodiments, amanipulator comprises one or more cell scrapers. Generally, manipulationlocations share many characteristics with imaging locations, asdescribed herein.

FIG. 2A depicts one illustrative embodiment of a cell culture incubator.In some embodiments, the incubator cabinet has a second imager (108).The second imaging location may be at or near the manipulation location(107). In some embodiments, the second imaging location and themanipulation location (107) are the same location. In some embodiments,a second imager (108) images the cells of cell culture vessels while thecells are manipulated by the manipulator (106). In some embodiments, thesecond imager is a bright-field microscope. In some embodiments, thesecond imager is a holographic microscope. In some embodiments, thefirst imager is a phase-contrast microscope.

FIG. 2B depicts one illustrative embodiment of a cell culture incubator.In some embodiments, the cell culture incubator has an imaging locationand a manipulation location that are the same location (105).

FIG. 3 depicts illustrative embodiments of further components of a cellculture incubator. Further components are any component of the incubatorthat is not listed in FIG. 1-2A or 2B. In some embodiments, a cellculture incubator contains barcoded cell culture vessels (109). Thus, insome embodiments, a cell culture incubator has a barcode scanner (110)located inside the internal chamber of the incubator cabinet. In someembodiments, the barcode reader communicates with a computer (111) torelay information about the cell culture vessel for which the barcodehas been scanned. In some cases, a barcode scanner may be affixed to anysurface of the internal chamber. For example, a barcode scanner can beaffixed to a wall of the internal chamber in close proximity to animaging location (105).

In some embodiments, the cell culture incubator contains at least oneprobe and/or at least one sensor (113) that measures environmentalconditions within the internal chamber. Examples of probes to measureenvironmental conditions include but are not limited to temperatureprobes, pressure probes, carbon dioxide (CO₂) sensors, oxygen (O₂)sensors and relative humidity sensors. In some embodiments, the at leastone probe and/or at least one sensor located within an instrumenthousing (112). The at least one probe and/or at least one sensor isconnected to a controller (114). In some embodiments, the controller(114) communicates with a computer (111). Additionally, the controller(114) may communicate with a fluid dispensing system (115). For example,if a CO₂ sensor indicates a low CO₂ level in the internal chamber, thecontroller (114) may direct the fluid dispensing system (116) to injectCO₂ gas into the internal chamber in order to increase the CO₂ level ofthe internal chamber.

FIG. 4 depicts schematics of illustrative embodiments of cell cultureincubators. In some embodiments, a manipulator (106) comprises one ormore cell scrapers (116), as shown in FIG. 4A. In some embodiments, amanipulator (106) comprises a plurality of cell scrapers (e.g., a bankof cell scrapers (117)), as shown in FIG. 4B. In some embodiments, theincubator comprises two manipulators (106 ₁ and 106 ₂), wherein eachmanipulator comprises a cell scraper (116), as shown in FIG. 4C. In someembodiments, the manipulator (106) is controlled by a controller (object(114) of FIG. 3) that is configured for programming of up to 360°rotation of the scraper tip/blade of the cell scraper (116), thusallowing for programming of scraping motion in coordination with linearmotion in the x-, y-. and z-axis, e.g., such that a constant angle ofattack is maintained between the scraper blade and the cells beingscraped, regardless of culture vessel well geometry.

FIG. 5 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 5A-5B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 5A-5B. In some embodiments, acell scraper comprises an opening (e.g., orifice) configured fortransporting (e.g., aspirating cells and/or cell culture media) (121),as shown in FIG. 5B. In some embodiments, a cell scraper comprises anopening connected to a channel (122), as shown in FIG. 5B. In someembodiments, a channel is integrated into a cell scraper (e.g., runningalong the inside or outside of a cell scraper handle and/or scrapingblade).

FIG. 6 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 6A-6B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 6A-6B. In some embodiments, acell scraper comprises an opening (e.g., orifice) configured fortransporting (e.g., aspirating, depositing, or aspirating anddepositing) cells and/or cell culture media (121), as shown in FIG. 6B.In some embodiments, a scraper comprises an opening connected to achannel (122), as shown in FIG. 6B. In some embodiments, a channel isanchored to a wall of the cell scraper.

FIG. 7 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 7A-7B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 7A-7B. In some embodiments, thescraping edge is positioned at an angle relative to the scraper handle.In some embodiments, a cell scraper comprises an opening (e.g., orifice)configured for transporting (e.g., aspirating, depositing, or aspiratingand depositing) cells and/or cell culture media (121), as shown in FIG.7B. In some embodiments, a cell scraper further comprises an openingconnected to a channel (122), as shown in FIG. 7B.

FIG. 8 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 8A-8B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 8A-8B. In some embodiments, thescraping edge is positioned at an angle relative to the scraper handle.In some embodiments, a cell scraper comprises an opening (e.g., orifice)configured for transporting (e.g., aspirating, depositing, or aspiratingand depositing) cells and/or cell culture media (121), as shown in FIG.8B. In some embodiments, a cell scraper comprises an opening connectedto a channel (122), as shown in FIG. 8B. In some embodiments the channelwalls are formed by the interior wall of the cell scraper, as shown inFIG. 8B.

FIG. 9 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 9A-9B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 9A-9B. In some embodiments, thescraping edge is positioned at an angle relative to the scraper handle.In some embodiments, a cell scraper comprises an opening (e.g., orifice)configured for transporting (e.g., aspirating, depositing, or aspiratingand depositing) cells and/or cell culture media (121), as shown in FIG.9B. In some embodiments, an opening further comprises a channel (122),as shown in FIG. 9B. In some embodiments the channel walls are formed bythe interior wall of the cell scraper, as shown in FIG. 9B.

FIG. 10 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 10A-10B. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 10A-10B. In some embodiments,the scraping edge is positioned at an angle relative to the scraperhandle. In some embodiments, a cell scraper comprises an opening (e.g.,orifice) configured for transporting (e.g., aspirating, depositing, oraspirating and depositing) cells and/or cell culture media (121), asshown in FIG. 10B. In some embodiments, cell scraper comprises anopening connected to a channel (122), as shown in FIG. 10B. In someembodiments the channel walls are formed by the interior wall of thecell scraper, as shown in FIG. 10B.

FIG. 11 depicts a schematic of illustrative embodiments of cellscrapers. In some embodiments, a cell scraper comprises a proximal end(118) and a distal end (119). In some embodiments, the distal end of thecell scraper comprises a scraping blade (e.g., a scraping edge) (120).In some embodiments, the scraping edge comprises multiple projections.

FIG. 12 depicts a schematic of illustrative embodiments of cellscrapers. In some embodiments, a cell scraper comprises a proximal end(118) and a distal end (119). In some embodiments, the distal end of thecell scraper comprises a scraping blade (e.g., a scraping edge) (120).In some embodiments, the scraping edge comprises multiple projections.In some embodiments, a cell scraper comprises an opening (e.g., orifice)configured for transporting (e.g., aspirating, depositing, or aspiratingand depositing) cells and/or cell culture media (121). In someembodiments, cell scraper comprises an opening connected to a channel(not shown). In some embodiments the channel walls are formed by theinterior wall of the cell scraper.

FIG. 13 depicts schematics of illustrative embodiments of cell scrapers.In some embodiments, a cell scraper comprises a proximal end (118) and adistal end (119), as shown in FIGS. 13A-13C. In some embodiments, thedistal end of the cell scraper comprises a scraping blade (e.g., ascraping edge) (120), as shown in FIGS. 13A-13C. In some embodiments,the scraping edge is positioned at an angle relative to the scraperhandle.

FIG. 14 depicts a schematic of a manipulator (106) comprising a cellscraper (116). In some embodiments, the manipulator (106) is controlledby a controller (not shown) that is configured for programming of up to360° rotational oscillation or linear oscillation of the scrapertip/blade of the cell scraper (116), thus allowing for programming ofscraping motion in coordination with linear motion in the x-, y-axis androtational motion around the z-axis, e.g., such that a constant angle ofattack is maintained between the scraper blade and the cells beingscraped, regardless of culture vessel well geometry.

FIG. 15 depicts schematics of illustrative embodiments of cell scrapers(116). In some embodiments, the distal end of the cell scraper comprisesa scraping blade (e.g., a scraping edge) (120), as shown in FIGS.15B-15C.

Automated Cell Culture

This document relates to incubators and methods for culturing,manipulating, and/or monitoring cells under controlled conditions (e.g.,under aseptic and/or sterile conditions). In some aspects, incubatorsand methods include automated components. In some aspects, incubatorsand methods are useful for long term cell culture (e.g., to grow andmaintain cells for recombinant protein expression or to grow and/ordifferentiate cells for therapeutic applications such as implantation).In some embodiments, cell cultures are grown within a culture vessel inan incubator described herein.

Culture Vessel

Cell culture vessels may be configured for culturing cells of differenttypes, including eukaryotic or prokaryotic cells. In some embodiments,cells are mammalian cells (e.g., human cells, canine cells, bovinecells, ovine cells, feline cells, or rodent cells such as rabbit, mouse,or rat cells). In some embodiments, cells are insect cells, avian cells,microbial cells (e.g., yeast cells such as Saccharomyces cerevisiae,Kluyveromyces lactis, or Pischia pastoris cells, or bacterial cells suchas Escherichia coli, Bacillus subtilis, or Corynebacterium cells),insect cells (e.g., Drosophila cells, or Sf9 or Sf21 cells), plant cells(e.g., algal cells) or cells of any other type.

In some embodiments, cells are cultured for producing natural products(e.g., taxols, pigments, fatty acids, biofuels, etc.). In someembodiments, cells are cultured to express recombinant products (e.g.,recombinant protein products such as antibodies, hormones, growthfactors, or other therapeutic peptides or proteins). In someembodiments, cells are expanded and/or differentiated for therapeuticuse such as implantation into a subject (e.g., a human subject) in orderto provide or supplement a cellular, tissue, or organ function that ismissing or defective in the subject.

In some embodiments, cells are from immortalized cell lines.Non-limiting examples of cell lines include human cells, for example,HeLa cells, prostate cancer cells (e.g., DU145, PC3 and/or Lncap cells),breast cancer cells (e.g., MCF-7, MDA-MB-438, and/or T47D cells), acutemyeloid leukemia cells (e.g., THP-1 cells), glioblastoma cells (e.g.,U87 cells), neuroblastoma cells (e.g., SHSY5Y cells), bone cancer cells(e.g., Saos-2 cells) and chronic myelogenous leukemia cells (e.g., KBM-7cells). In some embodiments, cell lines include primate cell lines,rodent cell lines (e.g., rat or mouse cell lines), canine cell lines,feline cell lines, Zebrafish cell lines, Xenopus cell lines, plant celllines, or any other cell. In some embodiments, cells are human 293 cells(e.g., 293-T or HEK 293 cells), murine 3T3 cells, Chinese hamster ovary(CHO) cells, CML T1 cells, or Jurkat cells.

In some embodiments, cells are primary cells, feeder cells, or stemcells. In some embodiments, cells are isolated from a subject (e.g., ahuman subject). In some embodiments, cells are primary cells isolatedfrom a tissue or a biopsy sample. In some embodiments, cells arehematopoietic cells. In some embodiments, cells are stem cells, e.g.,embryonic stem cells, mesenchymal stem cells, cancer stem cells, etc. Insome embodiments, cells are isolated from a tissue or organ (e.g., ahuman tissue or organ), including but not limited to, solid tissues andorgans. In some embodiments, cells can be isolated from placenta,umbilical cord, bone marrow, liver, blood, including cord blood, or anyother suitable tissue. In some embodiments, patient-specific cells areisolated from a patient for culture (e.g., for cell expansion andoptionally differentiation) and subsequent re-implantation into the samepatient or into a different patient. Accordingly, in some embodiments,cells grown in the incubators described herein may be used for allogenicor autogenic therapy. In some embodiments, cells grown in the incubatorsdisclosed herein may be genetically modified, expanded and reintroducedinto a patient for the purpose of providing an immunotherapy (e.g.,chimeric antigen receptor therapy (CAR-T), or delivery of CRISPR/Casmodified cells).

In some embodiments, a primary cell culture includes epithelial cells(e.g., corneal epithelial cells, mammary epithelial cells, etc.),fibroblasts, myoblasts (e.g., human skeletal myoblasts), keratinocytes,endothelial cells (e.g., microvascular endothelial cells), neural cells,smooth muscle cells, hematopoietic cells, placental cells, or acombination of two or more thereof.

In some embodiments, cells are recombinant cells (e.g., hybridoma cellsor cells that express one or more recombinant products). In someembodiments, cells are infected with one or more viruses.

Primary Cell Isolation

In some embodiments, cells are isolated from tissues or biologicalsamples for ex vivo culture in the incubators provided herein. In someembodiments, cells (e.g., white blood cells) are isolated from blood. Insome embodiments, cells are released from tissues or biological samplesusing physical and/or enzymatic disruption. In some embodiments, one ormore enzymes such as collagenase, trypsin, or proteinase are used todigest the extracellular matrix. In some embodiments, tissue orbiological samples are placed in culture medium (e.g., with or withoutphysical or enzymatic disruption), and cells that are released and thatgrow in the culture medium can be isolated for further culture.

Cell Culture

As used herein, cell culture refers to a procedure for maintainingand/or growing cells under controlled conditions (e.g., ex vivo). Insome embodiments, cells are cultured under conditions to promote cellgrowth and replication, conditions to promote expression of arecombinant product, conditions to promote differentiation (e.g., intoone or more tissue specific cell types), or a combination of two or morethereof.

In some embodiments, cell culture vessels are configured for culturingcells in suspension. In some embodiments, cell culture vessels areconfigured for culturing adherent cells. In some embodiments, cellculture vessels are configured for 2D or 3D cell culture. In someembodiments, cell culture vessels include one or more surfaces ormicro-carriers to support cell growth. In some embodiments, these arecoated with extracellular matrix components (e.g., collagen, fibrinand/or laminin components) to increase adhesion properties and provideother signals needed for growth and differentiation. In someembodiments, cell culture vessels include one or more synthetichydrogels such as polyacrylamide or polyethylene glycol (PEG) gels tosupport cell growth. In some embodiments, cell culture vessels include asolid support with embedded nutrients (e.g., a gel or agar, for examplefor certain bacterial or yeast cultures). In some embodiments, cellculture vessels include a liquid culture medium.

In some embodiments, cells are cultured in one of any suitable culturemedia. Different culture media having different ranges of pH, glucoseconcentration, growth factors, and other supplements can be used fordifferent cell types or for different applications. In some embodiments,custom cell culture media or commercially available cell culture mediasuch as Dulbecco's Modified Eagle Medium, Minimum Essential Medium, RPMImedium, HA or HAT medium, or other media available from LifeTechnologies or other commercial sources can be used. In someembodiments, cell culture media include serum (e.g., fetal bovine serum,bovine calf serum, equine serum, porcine serum, or other serum). In someembodiments, cell culture media are serum-free. In some embodiments,cell culture media include human platelet lysate (hPL). In someembodiments, cell culture media include one or more antibiotics (e.g.,actinomycin D, ampicillin, carbenicillin, cefotaxime, fosmidomycin,gentamycin, kanamycin, neomycin, penicillin, penicillin streptomycin,polymyxin B, streptomycin, tetracycline, or any other suitableantibiotic or any combination of two or more thereof). In someembodiments, cell culture media include one or more salts (e.g.,balanced salts, calcium chloride, sodium chloride, potassium chloride,magnesium chloride, etc.). In some embodiments, cell culture mediainclude sodium bicarbonate. In some embodiments, cell culture mediainclude one or more buffers (e.g., HEPES or other suitable buffer). Insome embodiments, one or more supplements are included. Non-limitingexamples of supplements include reducing agents (e.g.,2-mercaptoethanol), amino acids, cholesterol supplements, vitamins,transferrin, surfactants (e.g., non-ionic surfactants), CHO supplements,primary cell supplements, yeast solutions, or any combination of two ormore thereof. In some embodiments, one or more growth or differentiationfactors are added to cell culture media. Growth or differentiationfactors (e.g., WNT-family proteins, BMP-family proteins, IGF-familyproteins, etc.) can be added individually or in combination, e.g., as adifferentiation cocktail comprising different factors that bring aboutdifferentiation toward a particular lineage. Growth or differentiationfactors and other aspects of a liquid media can be added using automatedliquid handlers integrated within the incubators.

In some aspects, devices and methods described herein provide andmaintain appropriate temperature and gas mixtures for cell growth. Itshould be appreciated that cell growth conditions differ for differentcell types and that devices described herein can be programmed tomaintain different conditions. In some embodiments, conditions ofapproximately 37° C., and 5% CO₂ are used for mammalian cells.

In some embodiments, devices and methods described herein are used tomonitor or assay the culture media for nutrient depletion, changes inpH, changes in temperature, accumulation of apoptotic or necrotic cells,and/or cell density. For example a manipulator (106) may include sensorsthat monitor culture media. In some embodiments, devices and methodsdescribed herein are used to modify or change the culture media orconditions and/or to passage the cells when appropriate. In someembodiments, the devices and methods are automated (e.g., controlled bya controller (114) and/or computer (111), as shown in FIG. 3).

In some embodiments (e.g., for adherent cell cultures), culture mediacan be removed directly by aspiration and replaced with fresh media. Insome embodiments (e.g., for non-adherent/suspension cultures), mediachanges can involve centrifuging a cell culture, removing the oldculture media and replacing it with fresh media. In some embodiments,the centrifuge is located in the internal chamber of an incubator. Insome embodiments, culture vessels allow for continuous mediareplacement. In some embodiments, the incubators described herein mayinclude one or more components that can be used to process, replace,supply, and/or maintain different aspects of a culture media to supportcells. Incubators may include a reservoir containing waste media and/ora reservoir containing fresh media. Such reservoirs may be present(e.g., for temporary storage) within a refrigerator inside the incubatoror a refrigerated section of the incubator. In some embodiments, one ormore reservoirs are provided outside the incubators and piping isprovided into and out from the incubator space to supply or draw from aliquid handler units (e.g., liquid handle units having an aspirator) ortemporary reservoir within the incubator to facilitate cells feeding,media changes, and other related needs. For suspension cells, devicesmay be provided within the incubator to separate cells from waste media(e.g., centrifuge(s) to facilitate cell pelleting) to facilitateautomated media changes as part of an incubator provided herein. In someembodiments, the document provides a system comprising a cell cultureincubator connected to a computer, capable of automatically monitoringand adjusting cell culture conditions for optimal growth of the cellculture.

In some embodiments, cells are passaged within an incubator describedherein. In some embodiments, a cell culture is split and a subset of thecell culture is transferred to a fresh culture vessel for furthergrowth. In some embodiments (e.g., for adherent cell cultures), cellsare detached (e.g., mechanically, for example using gentle scraping,and/or enzymatically, for example using trypsin-EDTA or one or moreother enzymes) from a surface prior to being transferred to a freshculture vessel. In some embodiments (e.g., for suspension cellcultures), a small volume of a cell culture is transferred to a freshculture vessel.

In some embodiments, cell cultures are manipulated in other ways duringculture in incubators and vessels described herein. For example, cellcultures may be transfected with nucleic acids (e.g., DNA or RNA) orexposed to viral infection (e.g., using recombinant virus particles todeliver DNA or RNA).

Aseptic techniques can be used to prevent or minimize contamination ofcell cultures during growth and manipulation. In some embodimentsequipment (e.g., pipettes, fluid handling devices, manipulating devices,other automated or robotic devices, etc.) that is used for cell cultureis sterilized using an appropriate technique. Non-limiting techniquesinclude heat exposure (e.g., autoclaving), surface disinfection (e.g.,using alcohol, bleach, or other disinfectant), irradiation, and/orexposure to a disinfectant gas (e.g., ozone, hydrogen peroxide, etc.) asdescribed herein. In some embodiments, media is sterilized using anappropriate technique. Non-limiting techniques include heat exposure(e.g., autoclaving), antimicrobial/antiviral treatment, filtration,and/or irradiation.

In some embodiments, manipulations of cell cultures are performed underaseptic conditions, for example in an environment (e.g., within anincubator chamber) that has been disinfected and in which the air hasbeen filtered to remove potential contaminants.

In some embodiments, cell cultures are grown and maintained underGMP-compliant conditions, including using GMP-compliant media orGMP-compliant liquid handling equipment and performing methods inconjunction with standard operation procedures (SOPs).

In some embodiments, cell cultures can be monitored and/or evaluated todetect contamination. In some embodiments, contamination by cells from adifferent type of organism can be detected. In some embodiments,contamination of a mammalian cell culture by mycoplasma, bacteria,yeast, or viruses can be detected using any suitable technique. In someembodiments, cell culture contamination can be detected by assaying forchanges or for rates of change of one or more culture properties such aspH, turbidity, etc., that are characteristic of contamination (e.g., bybacteria or yeast) and not characteristic of the cells being grown inculture (e.g., mammalian cells). In some embodiments, one or moremolecular detection assays (e.g., PCR, ELISA, RNA labeling, or otherenzymatic techniques) or cell-based assays can be used to detectcontamination (e.g., mycoplasma, bacterial, yeast, viral, or othercontamination).

In some embodiments, cell cultures can be monitored and/or evaluated todetect contamination with cells of similar types (e.g., a human cellline contaminated by different human cells or by different mammaliancells). In some embodiments, cell cultures and their potentialcontamination can be evaluated using DNA sequencing or DNAfingerprinting (e.g., short tandem repeat-STR-fingerprinting), isoenzymeanalysis, human lymphocyte antigen (HLA) typing, chromosomal analysis,karyotyping, cell morphology, or other techniques.

In some embodiments, cells produced using devices and methods providedherein can be frozen to preserve them for later use and/or fortransport. In some embodiments, cells are mixed with a cryopreservationcomposition after growth and/or differentiation and prior to freezing. Acryopreservation composition can be added to a cell culture vessel orcells can be transferred from a cell culture vessel to acryopreservation vessel along with a cryopreservation composition.Non-limiting examples of cryoprotectants that can be included in acryopreservation composition include DMSO, glycerol, PEG, sucrose,trehalose, and dextrose. In some embodiments, a freezer may be presentin or in proximity with an incubator to facilitate freezing of cellsisolated from cell cultures.

Cell Culture Incubators:

This document relates to incubators and methods for culturing,manipulating, and/or monitoring cells under controlled conditions (e.g.,under aseptic and/or sterile conditions). In some embodiments, the cellculture incubators provided herein include an incubator cabinet definingan internal chamber for incubation of cells in one or more cell culturevessels, in which the internal chamber is configured to hold the one ormore cell culture vessels. In some cases, in addition to an internaldoor from the transfer chamber to the internal chamber, the incubatorsinclude at least one external door (e.g., 1, 2, 3, 4, or more externaldoors) opening from an external environment directly to the internalchamber, for example to provide alternative access to the internalchamber during periods of time when the incubator is not operational,e.g., during maintenance of the incubator. In some embodiments, theincubators include a storage location within the internal chamber forstoring one or more cell culture vessels. In some embodiments, a cellculture vessel transfer device is provided in the incubator for movingone or more cell culture vessels from a first imaging location to astorage location and/or from a storage location to an first imaginglocation.

In some embodiments, incubators or incubator cabinets provided hereinare rectangularly cuboidal in shape. In some embodiments incubators orincubator cabinets provided herein have a rectangular footprint in arange of 1 ft² to 16 ft². In some embodiments incubators or incubatorcabinets provided herein have a rectangular footprint of up to about 1ft², 2 ft², 3 ft², 4 ft², 5 ft², 6 ft², 7 ft², 8 ft², 9 ft², 10 ft², 11ft², 12 ft², 13 ft², 14 ft², 15 ft², or 16 ft². In some embodimentsincubators or incubator cabinets provided herein have a total chambervolume in a range of 1 ft³ to 100 ft³. In some embodiments incubators orincubator cabinets provided herein have a chamber volume of up to about1 ft³, 5 ft³, 10 ft³, 25 ft³, 50 ft³ or 100 ft³. In some embodimentsincubators or incubator cabinets provided herein have a rectangularfootprint in a range of 0.09 m² to 1.78 m². In some embodimentsincubators or incubator cabinets provided herein have a rectangularfootprint of up to about 0.1 m², 0.2 m², 0.3 m², 0.4 m², 0.5 m², 0.6 m²,0.7 m², 0.8 m², 0.9 m², 1.0 m², 1.1 m², 1.2 m², 1.3 m², 1.4 m², 1.5 m²,1.6 m², or 1.7 m². In some embodiments, incubators or incubator cabinetsprovided herein have a total chamber volume in a range of 0.03 m³ to 3m³. In some embodiments incubators or incubator cabinets provided hereinhave a chamber volume of up to about 0.03 m³, 0.1 m³, 0.3 m³, 1 m³, or 3m³.

Materials

In some embodiments, an incubator cabinet is single-walled. In someembodiments, an incubator is double-walled. In some embodiments,insulation material is provided between the double walls of an incubatorcabinet to control heat loss from the cabinet and facilitate temperaturecontrol in the cabinet. In some embodiments, the outer wall of anincubator cabinet includes a sheet metal, e.g., a 14-20 gauge coldrolled steel. In some embodiments, an inner wall (e.g., a chambersurface) of an incubator cabinet includes electro-polished stainlesssteel. In some embodiments, an inner wall (e.g., a chamber surface) ofan incubator cabinet includes corrosion resistant materials, such as,titanium, cobalt-chrome, tantalum, platinum, zirconium, niobium,stainless steel, and alloys thereof. However, in some embodiments, achamber surface of an incubator cabinet includes a polymeric materialsuch as polytetrafluoroethylene (PTFE), or a polymeric material knowunder the trade name of Parylene. In some embodiments, a chamber surfacemay have anti-microbial properties, such as copper or silver oranti-microbial compounds incorporated into a polymeric surface coating.

Monitoring Equipment

In some embodiments, the environment inside an incubator is controlledby a control system that may be configured to control the temperature,humidity, carbon dioxide, oxygen, and other gaseous components (e.g.,sterilization gases, such as, ozone, and hydrogen peroxide) inside theincubator (e.g., in one or more internal chambers). In some embodiments,a control system controls the environmental conditions (e.g.,temperature, humidity, carbon dioxide, oxygen and other gaseouscomponents) within each internal chamber separately. For example, inorder to protect sensitive mechanical, electronic and opticalcomponents, the humidity of an internal chamber may be maintained at alower level than an internal chamber having a storage location. In someembodiments, the incubator is further provided with a monitoring systemwith predefined sensors. Examples of monitoring devices include, but arenot limited, to oxygen monitors, carbon dioxide monitors, ozone gasdetectors, hydrogen peroxide monitors and multi gas monitors. Forexample, in some embodiments, an incubator advantageously includes aplurality of sensors responsive to different parameters relevant to cellgrowth, which may include temperature, air purity, contaminant levels,pH, humidity, N₂, CO₂, O₂, and light. By means of this monitoringsystem, parameters in the incubator can be measured using sensors forthe duration of a culture or process. In some embodiments, parametersmeasured by the sensors are transmitted by the monitoring system via aline to a computer-controlled monitoring and control system for furtherprocessing as discussed herein.

In some embodiments, an environmental monitoring system can be used inconjunction with an incubator described herein. In some embodiments, oneor more sensors that provide for the measurement of temperature, aircomposition (e.g., CO₂ concentration, O₂ concentration, etc.), and/orhumidity of the system can be associated with an incubator (e.g., fittedwithin an incubator cabinet). In some embodiments, one or more suchsensors can be incorporated as part of an incubator (e.g., attached to,integral to, or otherwise connected to an internal wall or door of theincubator). In some cases, one or more sensors can be positioned at anysuitable location(s) outside or inside an incubator cabinet (e.g.,within a transfer chamber and/or an internal chamber, for exampleattached to an internal wall, and/or upper or lower internal surface).

In some embodiments, a gas sensor is provided that can provide a readingin real time of the concentration of gas in contact with the sensor(e.g., gas in a cabinet, or ambient air) in percent, parts per million,or any other standard unit. Gas sensors for use in the methods andincubators provided herein include CO₂ sensors, O₂ sensors, N₂ sensors,ozone gas detectors, hydrogen peroxide monitors, multi gas monitors, andCO sensors. Such sensors are available from a number of commercialsources. In some cases, the environment of the incubator may bemodulated or controlled based upon the information provided by thesensors described herein. For example, the level of CO₂ in an incubatormay be increased upon indication from a CO₂ sensor that a lower thandesirable concentration of CO₂ is present in the incubator.

In some embodiments, one or more heating or cooling elements can beincorporated within the incubator (e.g., on an inner surface of thecabinet or door, and/or integrated within one or more of the wallsand/or the base of the cabinet) for purposes of controlling thetemperature within the incubator. In some embodiments, a heating elementcan be used for thawing liquids, for example, cell culture media orother reagents.

In some embodiments, one or more air or oxygen sources, carbon filters,and/or one or more humidification or dehumidification systems areconnected to the incubator and configured to control the level ofoxygen, carbon dioxide, and/or humidity within the incubator (e.g., inresponse to signals from the one or more sensors in or attached to theincubator). In some embodiments, one or more controllers are attached tothe sensors and other systems to control the internal environment of theincubator.

In some embodiments, an incubator can include one or more light sources(e.g., an incandescent bulb, LED, UV or other light source). These canbe placed within the incubator to illuminate regions within the cabinet.In some embodiments, the culture system operation is monitored using acamera or other light sensitive device that can be placed within oroutside the incubator. In some embodiments, the light source is asterilizing light source. For example, a UV lamp may be located withinthe transfer chamber and/or the interior chamber of the incubator.

In some embodiments, the incubator includes a transparent object (e.g.,window) that allows visible light or other light wavelengths from withinthe incubator to be detected by a camera or other light sensitive deviceplaced outside the incubator. In some embodiments, the inner surface ofthe transparent object can be wiped (e.g., from the inside of thecabinet) to prevent or remove condensation droplets that may accumulate(e.g., due to the humid air inside the incubator) on the inner surfaceand interfere with the monitoring of the system. In some embodiments,the surface can be wiped by a wiper that is automatically controlled bya controller.

Seals

In some embodiments, a culture cabinet includes windows, doors, oropenings that when closed are sealed to preserve sterility after theincubator cabinet has been sterilized. In some embodiments, each seal ofthe incubator cabinet is air tight up to a threshold level of pressure(e.g., up to 1 atm). In some embodiments, a gasket is provide to ensurea desired level of sealing capacity. In general, a “gasket” isunderstood as a mechanical seal that fills the space between twoobjects, generally to prevent leakage between the two objects whileunder compression. Gaskets are commonly produced by cutting from sheetmaterials, such as gasket paper, rubber, silicone, metal, cork, felt,neoprene, nitrile rubber, fiberglass, or a plastic polymer (such aspolychlorotrifluoro-ethylene). It is often desirable that a gasket bemade from a material that provides some degree of yielding such that itis able to deform and tightly fills the space it is designed for,including any slight irregularities. In some embodiments, gaskets can beused with an application of sealant directly to the gasket surface tofunction properly. In some embodiments, a gasket material can include aclosed-cell neoprene foam which is non-reactant with carbon dioxide orozone.

Transfer Devices

Incubators disclosed herein typically include one or more transferdevices for moving one or more items, e.g., from a first location to asecond location, within the incubators. In some embodiments, the one ormore items are one or more cell culture vessels. In other embodiments,the one or more items are useful for maintenance of one or more cellculture vessels and include, but are not limited to, pipettes,capillaries, liquids (e.g., cell culture medium), nutrients, and othermaterials. In some embodiments, a transfer device includes a roboticarm. In some embodiments, the robotic arm includes a platform within anincubator cabinet that may move along a rail or conveyor running invarious directions along an inner surface (e.g., inner wall, base, etc.)of incubator cabinet. In some embodiments, an incubator cabinet may beconfigured with more than one (e.g., 2, 3, 4, or 5, or more) roboticarms to increase the throughput of the instrument and to provideredundancy in the event that one of the robotic arms fail.

In some embodiments, a transfer device further includes a gripperassembly coupled to a robotic arm. In some embodiments, the gripperassembly includes one or more grippers mounted on the end of the roboticarm, each gripper having two or more (e.g., 3, 4, 5, or more) gripperfingers. In some embodiments, each of the gripper fingers on the roboticarm has a groove, friction plate, rubber pad, or other gripping surface.The gripping surface can allow the fingers to grip and transport varioustypes of containers (e.g., culture vessels) within the cabinets. In someembodiments, the robotic arm may have an absolute encoder either coupledto the gripper assembly, or the platform, or a separate absolute encoderfor each of the gripper assembly and/or the platform to determinewhether the robotic arm is in a position where it may be safely homed(e.g., returned to a resting or storage configuration and/or location ororigin of an operational coordinate system) without hitting anobstruction.

In some embodiments, because it may be desirable in certain situationsfor the reach of the robotic arm not to extend to some areas of theincubator cabinet, the robotic arm may instead reach these locations byinserting a container into or removing a container from a shuttle orconveyor belt, located, for example, on the incubator cabinet floor orother surface that moves along an axis (e.g., x-axis, y-axis) andprovides access to at least some of those locations to which the roboticarm cannot reach.

In some embodiments, an incubator cabinet is designed to be used inconjunction with an external assay or laboratory automation system. Forexample, in some embodiments, the incubator cabinet may have a doorhaving an opening large enough to allow the gripper arm to pivot outsideof the incubator cabinet with a sufficient reach for the fingers totransport culture vessels or other containers or components from atransport line of the laboratory automation system into the incubatorcabinet or transport external assay components into and/or out of theincubator cabinet.

In some embodiments, a robotic arm is designed to carry, among otherthings, culture vessels, in which case movements of the robotic arm arecontrolled to prevent jerking or accelerations of such vessels or othermovements which may cause the spilling of samples from the vessels. Insome embodiments, a robotic arm is designed to carry, among otherthings, culture vessels, in which case movements of the robotic arm arecontrolled to prevent movement of such vessels in ways which cause newlyplated cells to congregate/concentrate in specific areas of the culturevessel.

In some embodiments, because a robotic arm transports vessels or othercontainers between specific positions in the incubator cabinet, therobotic arm or other components of the incubator can be designed totrack precisely where the vessels or other containers are is located. Insome cases, in an incubator cabinet with which a robotic arm may beused, there are likely to be areas, such as where other components ofthe incubator cabinet or walls of the incubator cabinet are located, andthus where certain movements of the robotic arm may be limited. In thesecases, a homing mechanism can be used for each of various motors of thearms (e.g., x-motor, theta-motor and z-motor) to position properly therobotic arm to a known location after it is powered up or if a roboticarm collides with another object before resuming operation.

In some embodiments, an uninterruptible power supply (“UPS”) is attachedto or within the incubator cabinet, or contained with it, to allow foran orderly shut-down of incubator operations, including saving ofvarious automation and sample information and the completion of anytransport or transfer process that is underway (e.g., the transport of acontainer or vessel that is being carried by the robotic arm to itsdestination). The operator may be alerted to unauthorized opening of theincubator by an audible signal, a visual signal, an electronic signal(e.g., an email or a text message), or in some other manner.

In some embodiments, a sensor or other feature is provided to detectwhen one or more doors of an incubator are opened (e.g., when anincubator cabinet door, such as an external or internal door, isopened). Such features are useful because they allow operators to keeptrack of or be warned of any unscheduled or unauthorized openings of theincubator (e.g., the incubator cabinet) that could jeopardize sterility,spoil a production, compromise an assay or experiment, etc.

In some embodiments, a radiofrequency beacon or other signal source islocated within the incubator (e.g., within the incubator cabinet) thatcan be used to determine the location of one or more devices within theincubator cabinet (e.g., devices having sensors that can detect thesignal and use it to determine their location). In some embodiments, thedevices could have signal sources and the sensor(s) could be locatedwithin one or more of the chambers of an incubator cabinet (e.g.,located on an internal surface of an internal chamber).

In some embodiments, light signals or lasers (e.g., a grid of lasersignals) can be used to determine the location of one or more devices orcomponents within the incubator cabinet. Such information can becommunicated, e.g., wired or wirelessly, to an external computer ormonitoring station. The information can be used to control operation ofa transfer device, e.g., a robotic arm, within the incubator cabinet toensure that the transfer device can grab, manipulate, or maneuverdevices or items appropriately within the incubator cabinet.

In some embodiments, before containers or vessels are brought into anincubator cabinet, a user can select an automation system protocol basedon the particular containers, vessels, ingredients, or cells that arebeing inserted into the incubator cabinet. Relevant information relatedto the incubator and/or one or more incubator components, and the cellsbeing grown can be entered into a data system. For example, one or moreidentifiers such as barcodes (e.g., 1D or 2D barcodes) can be placed onthe container or vessel, and other significant information, such as, thetype of container, the contents of the container, what assays ormanipulations are to be performed on the sample in the container can bespecified. In some embodiments, information related to the incubatorsystem and/or cells can be contained in one or more barcodes, on aseparate data system, or a combination thereof. The user may also enterinformation that identifies the dimensionality (e.g., height, diameter)of the vessel or other container or the system itself can be configuredto determine the height or other dimensions of the vessel or othercontainer. Using this information, the robotic arm may be requested totransport a particular container, such as when an analytical module isready to perform an assay or other manipulation on cells grown in thevessels or has completed performing an assay or manipulation.

Computer and Control Equipment

The incubators provided herein include several components, includingsensors, environmental control systems, robotics, etc. which may operatetogether at the direction of a computer, processor, microcontroller orother controller. The components may include, for example, a transferdevice (e.g., robotic arm), a liquid handling devices, a delivery systemfor delivering culture vessels, or other components to or from theincubator cabinet, an environmental control system for controlling thetemperature and other environmental aspects of the incubator cabinet, adoor operation system, an imaging or detection system, and a cellculture assay system.

In some cases, operations such as controlling operations of a cellculture incubator and/or components provided therein or interfacingtherewith may be implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single component or distributed among multiple components. Suchprocessors may be implemented as integrated circuits, with one or moreprocessors in an integrated circuit component. A processor may beimplemented using circuitry in any suitable format.

A computer may be embodied in any of a number of forms, such as arack-mounted computer, a desktop computer, a laptop computer, or atablet computer. Additionally, a computer may be embedded in a devicenot generally regarded as a computer but with suitable processingcapabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable, mobile or fixed electronic device,including the incubator itself.

In some cases, a computer may have one or more input and output devices.These devices can be used, among other things, to present a userinterface. Examples of output devices that can be used to provide a userinterface include printers or display screens for visual presentation ofoutput and speakers or other sound generating devices for audiblepresentation of output. Examples of input devices that can be used for auser interface include keyboards, and pointing devices, such as mice,touch pads, and digitizing tablets. In other examples, a computer mayreceive input information through speech recognition or in other audibleformat, through visible gestures, through haptic input (e.g., includingvibrations, tactile and/or other forces), or any combination thereof.

One or more computers may be interconnected by one or more networks inany suitable form, including as a local area network or a wide areanetwork, such as an enterprise network or the Internet. Such networksmay be based on any suitable technology and may operate according to anysuitable protocol and may include wireless networks, wired networks, orfiber optic networks.

The various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Such software may bewritten using any of a number of suitable programming languages and/orprogramming or scripting tools, and may be compiled as executablemachine language code or intermediate code that is executed on aframework or virtual machine.

One or more algorithms for controlling methods or processes providedherein may be embodied as a readable storage medium (or multiplereadable media) (e.g., a computer memory, one or more floppy discs,compact discs (CD), optical discs, digital video disks (DVD), magnetictapes, flash memories, circuit configurations in Field Programmable GateArrays or other semiconductor devices, or other tangible storage medium)encoded with one or more programs that, when executed on one or morecomputers or other processors, perform methods that implement thevarious methods or processes described herein.

In some embodiments, a computer readable storage medium may retaininformation for a sufficient time to provide computer-executableinstructions in a non-transitory form. Such a computer readable storagemedium or media can be transportable, such that the program or programsstored thereon can be loaded onto one or more different computers orother processors to implement various aspects of the methods orprocesses described herein. As used herein, the term “computer-readablestorage medium” encompasses only a computer-readable medium that can beconsidered to be a manufacture (e.g., article of manufacture) or amachine. Alternatively or additionally, methods or processes describedherein may be embodied as a computer readable medium other than acomputer-readable storage medium, such as a propagating signal.

The terms “program” or “software” are used herein in a generic sense torefer to any type of code or set of executable instructions that can beemployed to program a computer or other processor to implement variousaspects of the methods or processes described herein. Additionally, itshould be appreciated that according to one aspect of this embodiment,one or more programs that when executed perform a method or processdescribed herein need not reside on a single computer or processor, butmay be distributed in a modular fashion amongst a number of differentcomputers or processors to implement various procedures or operations.

Executable instructions may be in many forms, such as program modules,executed by one or more computers or other devices. Generally, programmodules include routines, programs, objects, components, datastructures, etc. that perform particular tasks or implement particularabstract data types. Typically, the functionality of the program modulesmay be combined or distributed as desired in various embodiments.

Also, data structures may be stored in computer-readable media in anysuitable form. Non-limiting examples of data storage include structured,unstructured, localized, distributed, short-term and/or long termstorage. Non-limiting examples of protocols that can be used forcommunicating data include proprietary and/or industry standardprotocols (e.g., HTTP, HTML, XML, JSON, SQL, web services, text,spreadsheets, etc., or any combination thereof). For simplicity ofillustration, data structures may be shown to have fields that arerelated through location in the data structure. Such relationships maylikewise be achieved by assigning storage for the fields with locationsin a computer-readable medium that conveys relationship between thefields. However, any suitable mechanism may be used to establish arelationship between information in fields of a data structure,including through the use of pointers, tags, or other mechanisms thatestablish relationship between data elements.

In some embodiments, information related to the operation of theincubator (e.g., temperature, humidity, gas composition, images, cellculture conditions, etc., or any combination thereof) can be obtainedfrom one or more sensors associated with the incubator (e.g., locatedwithin the incubator cabinet, or located within the incubator butoutside the incubator cabinet), and can be stored in computer-readablemedia to provide information about conditions during a cell cultureincubation. In some embodiments, the readable media comprises adatabase. In some embodiments, said database contains data from a singleincubator. In some embodiments, said database contains data from aplurality of incubators. In some embodiments, data is stored in a mannerthat makes it tamper-proof. In some embodiments, all data generated bythe instrument (e.g., an incubator) is stored. In some embodiments, asubset of data is stored.

In some embodiments, the component (e.g., a computer) controls variousprocesses performed inside the incubator. For example, a computer maydirect control equipment (e.g., a manipulator, an imager, a fluidhandling system, etc.). In some embodiments, the computer controlsimaging of cell cultures, picking of cells, weeding of cells (e.g.,removal of cell clumps), monitoring of cell culture conditions,adjustment of cell culture conditions, tracking of cell culture vesselmovement within the incubator, and/or scheduling of any of the foregoingprocesses.

Cell Assays

In some embodiments, incubator cabinets provided herein are configuredwith a microscope or other imager or other device for purposes ofmonitoring cell growth, viability or other aspect of cells. In someembodiments, the microscope or imager is used in conjunction with anassay performed within the incubator cabinet, such as an image basedphenotypic screen or assay.

In certain embodiments, incubators provided herein are configured topermit one or more assays to be performed within an incubator cabinet orwithin a chamber operably connected to an incubator cabinet, e.g., aseparate assay chamber that is part of the incubator. In someembodiments, incubators provided herein are configured to permitperformance of a cell counting assay, a replication labeling assay, acell membrane integrity assay, a cellular ATP-based viability assay, amitochondrial reductase activity assay, a caspase activity assay, anAnnexin V staining assay, a DNA content assay, a DNA degradation assay,a nuclear fragmentation assay, or a combination thereof. Other exemplaryassays include BrdU, EdU, or H3-thymidine incorporation assays; DNAcontent assays using a nucleic acid dye, such as Hoechst Dye, DAPI,actinomycin D, 7-aminoactinomycin D, or propidium iodide; cellularmetabolism assays such as AlamarBlue, MTT, XTT, and CellTitre Glo;nuclear fragmentation assays; cytoplasmic histone associated DNAfragmentation assays; PARP cleavage assays; and, TUNEL staining assays.

Treatments and Experimental Interventions

In certain embodiments, incubators provided herein are configured topermit high-throughput screening (HTS) within an incubator cabinet. Insome embodiments, HTS refers to testing of up to, for example, 100,000compounds per day. In some embodiments, screening assays may be carriedout in a multi-well format, for example, a 96-well, 384-well format, or1,536-well format, and can be performed using automated protocols. Insuch high throughput assays, it is possible to screen several thousanddifferent compounds or compositions in a single day. In particular, eachwell of a microtiter plate can be used to run a separate assay against aselected test compound, or, if concentration or incubation time effectsare to be observed, a plurality of wells can contain test samples of asingle compound. It is possible to assay many plates per day; assayscreens for up to about 6,000, 20,000, 50,000, or more than 100,000different compounds are possible using the assays. Typically, HTSimplementations of the assays described herein involve the use ofautomation. In some embodiments, an integrated robot system thatincludes of one or more robotic arms transports assay microplatesbetween multiple assay stations for compound, cell, and/or reagentaddition, mixing, incubation, and finally readout or detection. In someaspects, an HTS assay may include preparing, incubating, and analyzingmany plates simultaneously, further speeding the data-collectionprocess.

In some embodiments, assays can include test cells, control cells, andone or more test compounds, e.g., 10, 100, 1000, 10,000, or more testcompounds. The cells and test agents can be arranged in one or morevessels in a manner suitable for assessing effect of the testcompound(s) on the cells. These assays can be performed within one ormore incubator cabinets of one or more incubators described herein.Typically, the vessels contain a suitable tissue culture medium, and thetest compounds are present in the tissue culture medium and may bedelivered to the culture medium within an incubator cabinet of anincubator provided herein in an automated fashion. A medium appropriatefor culturing a particular cell type can be selected for use. In someembodiments, a medium is free or essentially free of serum or tissueextracts, while in other embodiments such a component is present. Insome embodiments, cells are cultured on a plastic or glass surface.

The above aspects and embodiments may be employed in any suitablecombination, as the present invention is not limited in this respect.

It should be understood that aspects of the invention are describedherein with reference to certain illustrative embodiments and thefigures. The illustrative embodiments described herein are notnecessarily intended to show all aspects of the invention, but ratherare used to describe a few illustrative embodiments. Thus, aspects ofthe invention are not intended to be construed narrowly in view of theillustrative embodiments. In addition, it should be understood thataspects of the invention may be used alone or in any suitablecombination with other aspects of the invention.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the invention. Accordingly, the foregoing description anddrawings are by way of example only.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, e.g., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, e.g., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (e.g., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, e.g., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

1-32. (canceled)
 33. A cell scraper for use with a manipulator for manipulating cells in a cell culture vessel within in a cell culture incubator comprising: a handle portion comprising an elongate member extending from a proximal region that is connectable to a manipulator base and a distal region that comprises a scraping edge, a channel extending from the proximal region to the distal region, and an opening at the scraping edge in communication with the channel for transporting at least one of cells or cell culture media with respect to the cell culture vessel.
 34. The cell scraper according to claim 33, further comprising a contiguous structure comprising a scraping edge.
 35. The cell scraper according to claim 33, wherein each cell scraper comprises interconnected parts.
 36. The cell scraper according to claim 33, further comprising a handle having an interface for replaceably connecting a scraping edge assembly to the handle.
 37. The cell scraper according to claim 33, further comprising a scraper edge contactable with the surface of a cell culture vessel and configured for scraping cells adhering to the surface without substantially killing the cells.
 38. The cell scraper according to claim 37, further comprising a handle having an interface for replaceably connecting a scraping edge assembly to the handle.
 39. The cell scraper according to claim 37, further comprising a scraper edge contactable with the surface of a cell culture vessel and configured for scraping cells adhering to the surface without substantially killing the cells. 